Egypt. Acad. J. biolog. Sci., 2 (1): 39- 43 (2010) F.

Toxicology &Pest control

Email: egyptianacademic@yahoo.com ISSN: 2090 - 0791
Received: 25/4/2010 www.eajbs.eg.net

Biochemical and Toxicological studies on different field strains of cowpea aphid,

Aphis craccivora (Koch)

Azza I. Mohamed and Aziza, H. Mohamady.

Central Agricultural Pesticides Laboratory, Agricultural Research Center, Egypt
ABSTRACT
Field strains of copea Aphis craccivora selected (in October 2008) from different
Governorates (Behira, Gharbia, Sharkia and Faiyoum) were treated by different
groups of insecticides, to evaluate their efficacy against this pest and to study some
biochemical characters of these strains. The novel compound thiamethoxam
(neonicotinoid) was the most effective one followed by diafenthiuron (thiourea
compounds), carbosulfan (carbamate) and esfenvalerate (pyrethroid) in the all tested
Governments except Sharkia, diafenthiuron was the least effective one (LC50 232.80
ppm), while carbosulfan was the least effective one at Gharbia Governorate (LC50
109.7ppm.) Differences in protein content, protein patterns, esterase activity, alkaline
and acidic phosphatase were detected and differed from population to another.

INTRODUCTION 1990). Current work was to evaluate the

The sap sucking insects like aphids
constitute one of the major and important
economic pests of several different plants
in Egypt (Hassanein et al., 1971). Several
species of Aphis sp. damage a broad range
of vegetables like beans, cotton, tobacco,
tomato and ornamental plants (Greathead,
1986). Foliar applied insecticides are
recommended when insect populations
reach damaging levels but this is favorable
to the development of insect resistance to
many groups of insecticides (Georghiou,
1990). Aphids are difficult to be controlled
because of their mobility, tremendous
reproductive ability and resistance to many
synthetic pesticides (Van Lenteren, 1990),
however outbreaks have occurred in
association with insecticidal use for
controlling other pests (Slosser et al.,
1989). In general the novel compounds
including neonicotinoids, the newest major
class of insecticides have the potency and
systemic action of crop protection against
piercing sucking pests. They have low
toxicity to mammals, birds and fishes
(Foster et al., 2003 insecticide resistance
in agricultural pests is undesirable from
crop production point of view, some
insecticides present the opportunity to
investigate physiological mechanisms that
confer resistance beside minimum danger
to humans and natural enemies (Ishaaya.,
toxicity of used insecticides against A.
craccivora as one of the main pests, and to
study some biochemical characters of
these strains.
MATERIAL AND METHODS
Insect Strains:
1-Laboratory susceptible strain:
Susceptible strain of cow pea aphid, A.
craccivora (Koch) has been reared in
laboratory for 30 generations under
standard conditions at 22± 2 C0 and 70 ±5
R.H. and a photoperiod of 12:12 (Light
:Dark) hrs.
2- Field strains: Field strains of the aphid;
A. craccivora were collected from faba
bean fields of Behira, Gharbia, Sharkia,
and Faiyoum Governorates. Aphids were
collected in first October 2008. Field
populations were tested directly for
comparative studies of toxicological
activity of tested materials and for
biochemical studies.
Bioassay:
The leaf-dip bioassay was used
corresponded to that described by Moores
et al. (1996) with slight modifications.
Faba bean leaves were dipped in the
aqueous solution of any of the tested
insecticides for about 10 seconds and
allowed to dry on paper towel. They were
then placed upside down on an agar bed in
40 Azza I. Mohamed and Aziza, H. Mohamady
small petri-dish (60 mm diameter).Ten
apterous adults of A. craccivora were
placed on the treated leaf surface, while
leaves dipped in water serve as controls.
Three replicates batches of aphids (i.e, 30
insects) were used per each insecticide
concentration, and certain concentrations
(4-7) being used for each insecticide.
Petri-dishes containing aphids were kept in
rearing chamber temperature until
mortality was recorded.
Insecticides used:
Diafenthiuron (Polo 50% SC), Thiourea
insecticides Chemical name: 1-tert-butyl-
3-(2,6-di-isopropyl-4-phenoxyphenyl).
Carbosulfan (Marshal 25 % WP),
Carbamate insecticide.
Chemical name: 2,3 – dihydro - 2,2 -
dimethylbenzofuran-7-yl
(dibutylaminothio) methyl carbamate.
Esfenvalerate (Fast 20%…….) Pyrethroid
insecticide. Chemical name: [S-(R*,R*)]-
cyano(3-phenoxyphenyl)methyl4-chloro-
2-(1-ethylethyl)benzeneacetate.
Thiamethoxam: (Actara 25 % WP)
Neonicotinoid insecticide.
Chemical name: 3-(2-chloro-1,3-thiazol-5-
ylmethyl) -5- methy l-1, 3,5 -oxadiazinan-
4-ylidene (nitro) amine.
Data analysis:
LC50, slope values and fiducial
limits were estimated by using a software
package ″LD-P line″, copyright of Dr.
Ihab M.Bakr, Plant Protection Research
Institute.
Biochemical assay:
Adults of A. craccivora from
laboratory strain and field populations
were collected for investigation. The
whole insects were homogenized in 0.9%
Sodium Chloride solution by using hand
glass homogenizer on ice jacket. The
homogenate was centrifuged at 4000 rpm
for 15 min. at 4C0 and was stored at -20
C0. The supernatant obtained was used for
determination of total protein according to
Bradford (1976).
1) Determination of total enzymes
activity: Total esterase was determined
according to the method of Van Asperen
(1962). Acid and Alkaline phosphatise
were determined according to the method
described by Powel and Smith (1954).
2) Fractionation of total protein by
using denaturing SDS.PAGE: In a
solution of SDS and 2-mercaptoethanol,
proteins dissociated into subunits with rod
like shape, in which the diameter of the
rods is thought to be constant, while the
long axis varies according to the molecular
weight. The value can be determined by
comparing the electrophoretic mobility of
unknown protein with the mobility of
known standard protein marker, (Laemmli,
1970).
RESULTS AND DISSCUSION
Toxicity of different compounds aginst
aphids:-
The obtained data in table (1) clearly
indicated a great effect of the novel
compound thiamethoxam on all of the
tested aphids field strains. LC50 was 0.14,
0.64, 0.36, 0.44 and 0.75 ppm for
susceptible, Behira, Gharbia, Sharkia and
Faiyoum respectively.
Diafenthiuron which belongs to
novel insecticides; thiourea compounds
recorded the least effect on the Sharkia
population with the greatest LC50, 232.80
ppm among all insecticides used while it
recorded 18.30 ppm in Gharbia followed
by 23.60 and 27.93 ppm in Behira and
Faiyoum respectively, it may be due to the
high rate of IGI in Sharkia Governorate.
Carbosulfan (carbamate) insecticides had
moderate efficiency in all tested
Governorates except Gharbia (LC50,
109.70 ppm), while it was recorded 18.80,
78.04 and 46.07 ppm in Behira, Sharkia
and Faiyoum populations respectively. On
the other hand the pyrethroid compound,
esfenvalerate was the least effective one
on the susceptible strain and also on the
Behira and Faiyoum populations.
The present data proved that, the
neonicotinoid insecticides thiamethoxam
was the most effective one. These results
agree with Afzal et al. (2002);
Ulaganathan and Gupta (2004); Dewar et
al.(2004) and Mainfisch et al. (2001) who
demonstrated that from the Novel
compound, thiamethoxam exhibits,
exceptional systemic characteristics and
 Biochemical and Toxicological studies on different field strains of cowpea aphid 41

provides excellent control of a broad range
of commercially important pests, such as
aphids, jassids, whitefly and thrips.
Biochemical characters of different
aphid field strains:-
The protein content of susceptible
strain and each strain from four tested
Governorates was studies.
The obtained results (Table 2)
revealed that field strains had higher total
protein concentration than the susceptible
laboratory strain.
Faiyoum strain showed the highest
one with respect to total protein content
with 45mg/ mg protein while the strain
which had the lowest protein content, was
attained by Behira strain 17mg/ mg protein
followed by susceptible strain 15mg/ mg
protein. Also the total esterase content of
Faiyoum strain was reached to
2.77mM/mg protein followed by Sharkia,
Gharbia, and Behira strains (1.88, 1.61 and
1.36 mM/mg protein, respectively). The
esterase content of the susceptible strain
was 1.25 mM/mg protein.
Acid and Alkaline phosphatases
populations than the susceptible strains
might indicate that this lower level of
content Alkaline phosphatase accompanies
resistance rather cause it,i.e resistance
gene(s) might be associated with gene (s)
responsible for this low content (Amin,
1992). With respect to Acid phosphatase
activity, our data agreed with Farag
(1981), who stated that cypermethrin and
curacron resistant laboratory strains of S.
littoralis, had higher level of acid
phosphatase activity as compared to the
susceptible strain. El-Gendy et al. (1985),
stated that the higher acid phosphatase
activity in laboratory strain of the larvae of
S. littoralis might explain why that strain
was more tolerant than other strains for the
tested organophosphorus compounds.
Table (3) and Fig. (1) cleared that two
protein bands were appeared in field
populations and also in susceptible strain
(band number 27 and 29) whereas band
no. 18 appeared only in sus. strain with
Mw 75.78 KD. Also one band occurred
only in both susceptible strain and Sharkia
population (band no. 1 with Mw. 138.39),
while it did not appear in the others.
Table 3: Changes in protein patterns of A.
craccivora from four Governorates and
susceptible strain during October of the year
2008.
Band
No.
Rm
value
MW.
kd
Behira Faiyoum Sharkia Gharbia Sus.

activities were illustrated in table (2). Data 1

2
0.051
0.062
138.39
131.37
• •

showed that acid phosphatase activity in 3

4
0.118
0.125
131.03
126.55

the four field strains increased sharply 5

6
0.126
0.129
119.80
117.84 •
7 0.135 113.52
more than the susceptible strain (37.4 U/ 8 0.136 107.48
9 0.165 103.17
mg protein). It was 144.5, 102.1, 94.4, 10
11
0.166
0.171
96.61
91.23
•
•
and 87.9 U/ mg protein for Faiyoum, 12
13
0.176
0.256
89.91
87.31
•
•
Gharbia ,Sharkia and Behira, respectively, 14
15
0.296
0.303
85.25
81.32
• •
•
•

while alkaline phosphatase activity 16

17
0.332
0.372
78.31
77.50
• •
•
18 0.392 75.78 •
decreased in case of all tested populations 19 0.406 73.52 • •
20 0.443 70.13 •
than susceptible strain which recorded 21
22
0.493
0.552
68.81
66.30
•
•
238.90 U/ mg protein while other 23
24
0.601
0.653
63.91
60.30
•
•
•
•
populations recorded 198, 173, 163.4, and 25
26
0.702
0.758
58.50
56.10
•

189.6 U/ mg protein for Faiyoum, Gharbia 27

28
0.793
0.817
49.30
36.20
• •
•
•
•
• •

29 0.858 35.21 • • • • •
,Sharkia and Behira, respectively. 30 0.908 34.20 • •
31 0.920 33.25 • •
The lower level of Alkaline 32
33
0.950
0.970
30.23
28.13 •
phosphatase of aphid field strains than 34
35
0.990
0.999
26.20
20.20
susceptible strain in the present work agree Rm: Relative mobility. Mw: Molecular weight.
with Abd-Hafez (1978) and Farag (1978) Kd: kilo Dalton.
who reported that organophosphorus
resistant laboratory strain of S. littoralis
have much lower Alk-phos. activity than
the susceptible strain. The presence of
lower level of Alk-pase content in field
 42 Azza I. Mohamed and Aziza, H. Mohamady
Molecular weight
M. B. F. Sh. G. Su.
Fig. 1: SDS-polyacrilamide gel electrophoresis of
total protein patterns in the different field
strains and susceptible strain of Aphis
craccivora.
M.: Marker, B.: Behira, F.: Faiyoum, Sh.:
Sharkia,. G.:Gharbia, Su: susceptible.
Faiyoum and Sharkia populations
recorded the highest number of protein
bands (11 and 10 bands, respectively),
these results agree with the values of LC50
where they had the highest LC50’samong
the other populations and also agree with
Hama and Hosoda (1988). They found
good relationship between protein content
and carboxylesterase activity of A.
gossypii. Also, Shuai and Wang (2005)
stated that, resistance strain of Myzssyus
persicae developed obvious resistance to
alphamethrin, as resistance developed
enzyme protein content increased
significant correlation between LD50’s
values and enzymes protein content, as
well as esterase activity.
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ARABIC SUMMARY

‫دراسات بيوكيميائية وتوكسيكولوجية علي سالالت حقلية مختلفة لمن الفول‬

‫ عزيزة حسن محمدي‬- ‫عزة إسماعيل محمد‬

‫ الدقي‬- ‫ مركز البحوث الزراعية‬-‫المعمل المركزي للمبيدات‬

‫ت م دراس ة ت أثير أرب ع مج اميع مختلف ة م ن المبي دات وھ ي م ن مجموع ات الثيويوري ا والكرباماتي ة و‬
‫ م ن محافظ ات‬٢٠٠٨ ‫النيوكوتونيدية و البيروثرودية علي بع ض الس الالت الحقلي ة والت ي ت م تجمعھ ا ف ي أكت وبر‬
‫ حيث أستخدمت ساللة محافظة البحيرة ممثلة لغرب الدلتا والغربي ة ممثل ة لوس ط ال دلتا ومحافظ ة الش رقية‬.‫مختلفة‬
‫ممثل ة لش رق ال دلتا والفي وم ممثل ة لجن وب ال دلتا لتقي يم فاعلي ة ھ ذه المبي دات عل ى ھ ذه األف ة ولدراس ة الص فات‬
.‫البيوكيميائية لھذه السالالت‬
diafenthiuron ‫ من المبيدات الجديدة أعلى فاعلية يلي ة مرك ب‬thiamethoxam ‫وقد أعطي مركب‬
‫ م ن المبي دات‬esfenvalerate ‫ م ن المبي دات الكرباماتي ة ث م‬carbosulfan ‫م ن مركب ات الثيويوري ا ث م‬
‫ أق ل فاعلي ة مقارن ة‬diafenthiuron ‫البيروثرويدية لكل المحافظات ماع دا محافظ ة الش رقية حي ث أظھ ر مرك ب‬
‫ أق ل فاعلي ة‬carbosulfan ‫ جزء فى المليون بينما أعطي مركب‬232.80 LC50 ‫ فقد كانت قيمة‬LC50 ‫بقيم الـ‬
‫ وق د وج دت أختالف ات ف ي‬.‫ ج زء ف ي الملي ون‬١٠٩.٧ ‫ف ي محافظ ة الغربي ة حي ث ك ان التركي ز النص ف ممي ت‬
‫ وقد لوحظ أيضا‬. ‫المحتوى الكلي للبروتين وكذلك الحزم البروتينية عند تفريد البروتين بجھاز األلكتروفوريسيس‬
.‫أختالف في نشاط انزيم االستريزيز والفوسفاتيز القاعدي و الحامضي لھذه األفة من محافظة ألخرى‬